My today’s blog is on a very interesting review article by Dr. RA Weiss on the discovery of endogenous retroviruses (ERVs). These were discovered in late 1960s and early 1970s. Three types of ERVs were discovered around the same time. These were avian leucosis virus in the domestic fowl, murine leukaemia virus and murine mammary tumor virus in the laboratory mice. Retroviruses in general can be classified into two types: those having simple genome and those with complex genome. Only the simple retroviruses have become endogenous in their hosts with the exception of spumaviruses (have complex genome).
Retroviruses and the proviral hypothesis-
Retroviral diseases were recognized very early in 20th century, although the name retrovirus was coined only in 1974. In 1904, Vallée and Carré showed that equine anemia was infectiously transmitted by a filtrate and we now know that the etiologic agent is a lentivirus. In 1961, it was shown that Rous Sarcoma virus (RSV) particles contain RNA. Howard Temin observed that cells transformed by RSV maintained stable properties through many mitoses. Based on this inheritability of virus transformed phenotypes even in the absence of viral replication, he postulated that RSV genome makes a DNA copy in the infected cell. This DNA copy is then integrated into the host chromosomal DNA. He called this concept as DNA proviral hypothesis. Dr. Andre Lwoff, who had won a noble prize for the discovery of the prophage and lysogeny, has suggested integration of DNA virus, polyoma virus, into the host genome. The concept of integration of DNA tumor viruses genomes in transformed somatic cells was debated and demonstrated in 1968 by Sambrook et al. In spite of all these advances in the understanding of these concepts, the idea of integration of DNA genomes of RNA tumor viruses into the germ-line of healthy animals was not considered.
Endogenous Avian leucosis virus (ALV)
ALV is an alpha retrovirus and it replicates in chick embryo fibroblasts but does not transform them. It is closely related to RSV. However, RSV carries the src oncogene and transforms fibroblasts. The genome organization of these viruses is:
ALV: 5' LTR-gag-pol-env-LTR 3'
RSV (Bryan): 5' LTR-gag-pol-src-LTR 3'
RSV (Prague): 5' LTR-gag-pol-env-src-LTR 3'
The Bryan strain of RSV is defective for replication as src gene is substituted for env gene. Defective Bryan strain can be rescued by ALV which supplies the missing env glycoproteins. Thus, ALV was called a helper virus.
In 1960s, avian leucosis had become a problem in egg-lying hens and thus to screen for leucosis a serological test was devised for group specific antigen (gag) which was common to all ALV serotypes. This test was based on complement fixation and was called COFAL test. It was later observed that the test was not sufficiently specific as some uninfected chickens gave positive results. Furthermore, virus like particles and gag like antigens were detected from ALV-free chicken tissue. Then Payne and Chubb demonstrated that gag-related antigen was inherited as a dominant Mendalian gene in crosses between gag positive and gag negative chicken. Thus, the question arose that whether the endogenous antigen was encoded by a latent retroviral genome or whether it represented a normal host protein with a cross reacting epitope.
Dr. Weiss had observed during his PhD thesis that fibroblast cultures of some chick embryos but not the others, allowed release of infectious Bryan RSV in the absence of “helper” ALV. Dr. Weiss also observed that the envelope of helper free RSV was novel in its receptor specificity and neutralization properties. Similar results were also produced by other labs. Thus, it became evident that some normal chick cells could provide the missing env protein to Bryan RSV. Based on all these observations, Dr. RA Weiss published his work and suggested existence of an integrated retrovirus in normal embryo cells.
Two lines of evidence namely, Mendelian inheritance of gag-related antigen and complementation of an env defective strain of RSV provided a clue that something related to a retrovirus existed in the normal chick embryo cells. So the next step was to determine whether Env complementation and Gag expression were inherited concomitantly. Using inbred chickens, F1 hybrids and back-crosses, the researchers found that both phenotypes were indeed inherited according to Mendel's first law and that they segregated together as a single locus. In 1970, Dr Weiss and Dr. Peter Vogt provided evidence that treatment of normal chicken cells with a variety of activating agents such as ionizing radiations or carcinogens, stimulated release of virus. The authors further used reverse transcriptase activity to measure release of virus particles. Later, based on southern hybridization many proviral copies were found to be present in most chicken breeds. These individual proviral loci were characterized and mapped, many represented incomplete or defective genome.
Dr. Weiss was further interested to know if the chicken ERV was a recent introduction into domestic fowl, or whether it was present in the ancestor species, the red jungle fowl. In 1970, he made a field trip to Malaysia and lived with tribesmen (orang asli) in the Pahang jungle who knew how to trap these birds, in order to take blood samples and to collect eggs for cell culture. The red jungle fowl carried endogenous ALV. He and his team later found that the three other extant species of the same genus, Gallus, did not possess endogenous ALV. Apparently this ERV colonized the chicken germ-line after speciation but before domestication.
Astrin et al identified a rooster that lacked any integrated provirus and a line of chickens was eventually bred from this bird. This showed that viral genomes were not essential for host functions. However, these chickens do carry a second family of ERV called endogenous avian virus (EAV) although they are not infectious. EAV sequences are present in DNA of all species of Gallus and therefore have a more ancient origin.
A third group of avian retroviruses include reticuloendotheliosis virus of turkey.
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